Cloning,Expression Of KLF8,KLF10,KLF13,and Study On Promoter Activity Of C/EBPs In Dalian Purple Sea Urchin,Strongylocentrotus Nudus

The Dalian purple sea urchin,Strongylocentrotus nudus is an important economic species in Chinese aquaculture because of the rich nutrition of various unsaturated fatty acids in their gonad.However,the mechanisms of fatty acid synthesis and lipid formation have not yet been studied in Strongylocentrotus nudus.Krüppel-like factors(KLFs)and CCAAT/enhancer binding proteins(C/EBPs)has been elucidated to regulate lipid formation in a complex network of many TFs.KLFs,a zinc finger TF family consisting of 17 members,of which five members including KLF15,KLF4,KLF5,KLF6 and KLF9 take part in the positive regulation of adipogenesis,two members(KLF2 and KLF3)are in the negative way.C/EBPs are a family of leucine-zipper TFs and play a key role in adipogenesis.So far,six members(C/EBP?,C/EBP?,C/EBP?,C/EBP?,C/EBP? and C/EBP?)of the family have been identified from human.Therefore,to elucidate roles of Sn KLFs genes and the regulatory mechanisms of Sn C/EBPs genes transcription in Strongylocentrotus nudus,which will be beneficial to a better understanding of the mechanisms in gonadal lipid formation.In this study,the genes related to lipid formation were screened in transcriptome,gene specific primers were designed and combining the technology of real-time fluorescent quantitative PCR,the technology of Genome Walking,constructing overexpression vectors and truncated promoter vectors,the Dual-Luciferase Reporter Assay System and bioinformatics analysis.we obtained full-length c DNA sequence of Sn KLF8,Sn KLF10,Sn KLF13 and three promoter sequences of Sn C/EBPs from Strongylocentrotus nudus,analyzed their sequences characteristics and expression profile,and explored their function in lipid formation,finally obtained the following results:(1)The Sn KLF8 c DNA is 3945 bp in length with an ORF of 1164 bp encoding 387 amino acids(aa),a 97 bp 5? UTR and a 2684 bp 3? UTR.The Sn KLF10 c DNA was 3526 bp in length with an ORF of 1476 bp encoding 491 amino acids(aa),a 138 bp 5`-UTR and a 1912 bp 3`-UTR.The Sn KLF13 c DNA is 3756 bp in length with an ORF of 849 bp encoding 282 aa,a 422 bp 5? UTR and a 2485 bp 3? UTR.Three highly conserved Zn F_C2H2 domains located at Sn KLF8,Sn KLF10 and Sn KLF13 C-terminus were found by analysis of domain.(2)The Sn C/EBP?,Sn C/EBP? and Sn C/EBP? c DNA with an ORF encoding complete amino acids(aa)were identified from Strongylocentrotus nudus.And Sn C/EBP?,Sn C/EBP? and Sn C/EBP? proteins contained a BRLZ domain located at C-terminus were found by analysis of domain.Furthermore,the promoter sequences of Sn C/EBP?(1154 bp),Sn C/EBP?(1494 bp)and Sn C/EBP?(823 bp)were cloned.(3)Sn KLF8,Sn KLF10,Sn KLF13 and Sn C/EBPs were expressed predominantly in male and female gonads,and the m RNA expression levels were found to be different in second-instar and third-instar gonads.(4)The p EGFP-KLF8 and p EGFP-KLF8 overexpression all led to a lower promoter activity of C/EBP?-1,C/EBP?-1 and C/EBP?-1 relative to their p EGFP-control,which showed that Sn KLF8 and Sn KLF10 suppress Sn C/EBPs genes transcription.However,Overexpression of p EGFP-KLF13 leads to a higher promoter activity of C/EBP?-1 relative to the p EGFP-control,whereas lower promoter activity of C/EBP?-1 and C/EBP?-1 relative to their p EGFP-control,which showed that Sn KLF13 was able to promote Sn C/EBP? gene transcription,while suppress Sn C/EBP? and Sn C/EBP? genes transcription.(5)The TFBSs at Sn C/EBP?,Sn C/EBP? and Sn C/EBP? were predicted by bioinformatics analysis,which further show that Sn C/EBPs are potential target genes for Sn KLFs transcription factors.(6)The Dual-Luciferase Reporter Assay System showed that a potential core promoter region was located between nucleotides-204 and-16 in Sn C/EBP?,two potential core promoter region were located between nucleotides-1396 to-1179(core promoter region 1) and nucleotides-742 to-525(core promoter region 2)in Sn C/EBP?,a potential enhanced promoter region located in nucleotides 164 and 609 in Sn C/EBP?.The above results implied that Sn KLF8,Sn KLF10,Sn KLF13 and C/EBPs play a potential role in lipid formation,and which play a potential role in this biological process by regulating Sn C/EBPs genes transcription.The results will lay a foundation for the further researches of Sn KLF8,Sn KLF10,Sn KLF13 and C/EBPs genes roles in invertebrates and give a new insight into the regulatory mechanisms of Sn C/EBPs genes transcription.